The four established or putative sphingolipid activator proteins derive from a large precursor protein encoded by a single gene. In addition to generating the four sphingolipid activator proteins, the precursor protein is suspected of having functions of its own, as, for example, a lipid binding/transport protein or a neurotrophic factor. The gene also appears to encode the Sertoli cell major sulfated glycoprotein. Sequence similarities have been noted with many other proteins of diverse functions. One patient and a fetus in a single family with a complete defect of this gene due to a mutation in the initiation codon exhibited complex pathological and biochemical abnormalities. Mutant mice homozygous for an inactivated gene of the sphingolipid activator protein precursor exhibit two distinct clinical phenotypes-neonatally fatal and later-onset. The latter develop rapidly progressive neurological signs around 20 days and die by 35-38 days. At 30 days, severe hypomyelination and periodic acid-Schiff-positive materials throughout the nervous system and in abnormal cells in the liver and spleen are the main pathology. Most prominently lactosylceramide, and additionally ceramide, glucosylceramide, galactosylceramide, sulfatide, and globotriaosylceramide are abnormally increased in the brain, liver, kidney, and their catabolism abnormally slow in cultured fibroblasts. Brain gangliosides are generally increased, particularly the monosialogangliosides. The clinical, pathological and biochemical phenotype closely resembles that of the human disease. This model not only allows further clarification of the physiological functions of the four individual sphingolipid activator proteins but also should be useful to explore putative functions of the precursor protein.

No abstract

Nonredox type 5-lipoxygenase (5-LO) inhibitors, such as ZM 230487, its methyl analogue ZD 2138, or the Merck compound L-739,010, suppress cellular leukotriene synthesis of ionophore stimulated granulocytes with IC 50 values of about 50 nM. However, in cell homogenates or in preparations of purified enzyme, up to 150-fold higher concentrations are required for similar inhibition of 5-LO activity. This loss of 5-LO inhibition in cell homogenates was reversed by addition of glutathione or dithiothreitol, which increased the inhibitory potency of ZM 230487 or L-739,010 by about 100 to 150-fold so that 5-LO inhibition was comparable with that of intact cells. In the presence of thiols, addition of hydroperoxide [13(S)-HpODE], glutathioneperoxidase inhibition by iodacetate or selenium-deficiency lead to impaired 5-LO inhibition by ZM 230487 in cell homogenates. Moreover, addition of glutathione peroxidase was required for efficient inhibition of purified human 5-LO by ZM 230487. The data suggest that low hydroperoxide concentrations are important for efficient 5-LO inhibition by ZM 230487. The kinetic analysis revealed a noncompetitive inhibition of 5-LO by ZM 230487 at low hydroperoxide levels, whereas it acted as a competitive inhibitor with low affinity under nonreducing conditions in granulocyte homogenates. No such redox-dependent effects were observed with the 5-LO inhibitor BWA4C, the 5-LO activating protein-inhibitor MK-886 or the pentacyclic triterpene acetyl-11-keto-␤-boswellic acid. These data suggest that physiological conditions associated with oxidative stress and increased peroxide levels lead to impaired efficacy of nonredox type 5-LO inhibitors like ZM 230487 or L-739,010. This could explain the reported lack of activity of this class of 5-LO inhibitors in chronic inflammatory processes.

In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAPprecursor proteins with or without 2-and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by ␤-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursordeficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipidloaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP-B can function as sulfatide/ globotriaosylceramide activator.

The lysosomal degradation of sphingolipids with short oligosaccharide chains depends on small glycosylated non-enzymatic sphingolipid activator proteins (SAPs, saposins). Four of the five known SAPs, SAP-A, -B, -C and -D, are derived by proteolytic processing from a common precursor protein (SAP-precursor) that is encoded by a gene on chromosome 10 consisting of 15 exons and 14 introns. SAP-B is a non-specific glycolipid binding protein that stimulates in vitro the hydrolysis of about 20 glycolipids by different enzymes. In vivo SAP-B stimulates in particular the degradation of sulphatides by arylsulphatase A. So far, four different point mutations have been identified on the SAP-B domain of the SAP-precursor gene. The mutations result in a loss of mature SAP-B, causing the lysosomal accumulation of sulphatides and other sphingolipids, resulting in variant forms of metachromatic leukodystrophy (MLD). Here we report on a patient with SAP-B deficiency that is caused by a new homoallelic point mutation that has been identified by mRNA and DNA analysis. A 643A > C transversion results in the exchange of asparagine 215 to histidine and eliminates the single glycosylation site of SAP-B. Metabolic labelling experiments showed that the mutation had no effect on the intracellular transport of the encoded precursor to the acidic compartments and its maturation in the patient's cells. All four SAPs (SAP-A to SAP-D) were detectable by immunochemical methods. SAP-B in the patient's cells was found to be slightly less stable than the protein in normal cells and corresponded in size to the deglycosylated form of the wild-type SAP-B. Feeding studies with non-glycosylated SAP-precursor, generating non-glycosylated SAP-B, showed that the loss of the carbohydrate chain reduced the intracellular activity of the protein significantly. The additional structural change of the patient's SAP-B, caused by the change of amino acid 215 from asparagine to histidine, presumably resulted in an almost completely inactive protein.